Fig. 6

Forced LDB1 targeting to PAX3 bound elements induces skeletal myogenesis. a Schematic representation of PAX3 interactions with the HMT complex and LDB1. CXXC1 and PAGR1A subunits are present in the HMT complex responsible for deposition of H4K4me3 (SET1A complex) and H4K4me1 (MLL3/4 complex), respectively. b Schematic representation of PAX3 (WT), PAX3 lacking the transactivation domain (ΔTAD) and the fusion proteins involving LDB1, ASH2L, CXXC1, and PAGR1A. Size is not indicative of the real protein length. Pax3 domains: PD, paired domain; HD, homeodomain; TAD, transactivation domain. c The PAX3 TAD is required for LDB1 recruitment. ChIP-qPCR analysis of LDB1 recruitment at the Myf5 −111 kb locus in 1-day WT- and ΔTAD-induced (+) and non-induced (−) cells. Myf5 −257 kb: negative control. Graph represents mean + s.e.m. from n = 4 independent experiments. Student’s t-test **p < 0.01. d Analysis of the skeletal myogenic activity of WT, ΔTAD, ΔTAD-LDB1, ΔTAD-ASH2L, ΔTAD-CXXC1, and ΔTAD-PAGR1A. Transgenes were induced for 6-day following the scheme presented in Fig. 1a. Non-induced cells are used as negative control of myogenic commitment. Immunostaining: MYOG antibody (yellow) DAPI (Nuclei - blue). Images are representative of independent biological replicates (n = 3). Scale bar: 100 µm. e Gene expression analysis of Pax3 targets following 1-day induction of WT, ΔTAD, ΔTAD-LDB1 and ΔTAD-ASH2L. Graph represents mean + s.e.m. of n ≥ 3 independent biological replicates. Student’s t-test *p < 0.05, **p < 0.01, ***p < 0.001. f Long-range interactions at the Cdon, Dbx1, Myf5, and Prrx1 loci in 1-day ΔTAD-LDB1-induced cultures. Normalized contact maps based on sequencing depth are visualized as ratio relative to the non-induced cells. Position of PAX3 peaks, genes, and chromosome coordinates are displayed below the matrix. Scale: maxrange = 0.2. Arcs indicate looping interactions identified by FitHiChIP. g k-means clustering of H3K4me1 ChIP-seq data from 1-day induced (+) and non-induced (−) WT, ΔTAD, and ΔTAD-LDB1 cultures. Density Tag Map for H3K4me1 is centered on 3780 PAX3 peaks ±3 kb. Cluster 3 shows an increase in H3K4me1 deposition following WT and ΔTAD-LDB1 induction. Cluster 1: 457 peaks; Cluster 2: 1420 peaks; Cluster 3: 1903 peaks. h Distribution of H3K4me1 ChIP-seq reads across the PAX3 peak center ±3 kb for cluster 3 shown in panel g. Source data are provided as a Source Data file